Panel Design and Fluorochrome Selection Flashcards

What is the purpose of a fluorescence minus one (FMO) control in a multicolor flow cytometry panel?

Answer: To define accurate gate boundaries by capturing spillover spread from all other fluorochromes

FMO controls contain every fluorochrome except the one being gated, so the signal seen in that channel represents only the spread from other fluorochromes, enabling accurate gate placement.

Which of the following best describes 'spreading error' in multicolor flow cytometry?

Answer: Increased variance in fluorescence measurements caused by spectral spillover and its mathematical compensation

Spreading error is the additional variance introduced when compensation is applied to correct for spillover; it increases noise and can reduce the ability to resolve closely spaced populations.

When designing a panel on an instrument with limited laser lines, which strategy best maximizes the number of parameters measured?

Answer: Selecting fluorochromes with widely separated emission peaks on each laser

Choosing fluorochromes whose emission peaks are widely spaced minimizes spectral overlap, allowing more independent detection channels to be used per laser line.

What characteristic makes PE (phycoerythrin) one of the most valuable fluorochromes in flow cytometry panels?

Answer: Its extremely high quantum yield and molar absorptivity resulting in exceptional brightness

PE's very high quantum yield combined with a large absorption cross-section makes it one of the brightest fluorochromes available, ideal for detecting low-abundance targets.

Which of the following is a recognized disadvantage of using tandem dyes in flow cytometry experiments?

Answer: They show lot-to-lot variability and can degrade, producing incomplete energy transfer artifacts

Tandem dyes are susceptible to dye degradation and lot-to-lot spectral variability because partial breakdown of the FRET pair causes donor emission bleed-through, complicating compensation.

In spectral flow cytometry, what replaces traditional bandpass filters for signal detection?

Answer: Prisms or diffraction gratings that disperse light across an array of detectors

Spectral cytometers use optical elements such as prisms or gratings to spread emission light across a detector array, capturing the complete emission spectrum of each event.

What does 'unmixing' refer to in spectral flow cytometry data analysis?

Answer: A mathematical process that deconvolves overlapping spectra to quantify each fluorochrome's individual contribution

Unmixing uses reference spectra for each fluorochrome and a least-squares algorithm to separate the contributions of all fluorochromes from the composite signal detected on each event.